Rhesus monkeys (Macaca mulatta) infected with Plasmodium cynomolgi were used to test the hypothesis that the parasites which cause relapse are identical to those that cause the initial bloodstream infection. Primary sporozoite-induced infections were produced using Anopheles stephensi mosquitoes that had been fed on a splenectomized rhesus monkey with P. cynomolgi infection. The parasitemia occurred 10-14 days after sporozoite inoculations, and was treated with chloroquine after obtaining blood specimens for cryopreservation and filter paper blots. One month later, at the time of relapse, blood specimens were obtained again and extracted for DNA. The polymerase chain reaction (PCR) was then used to amplify variable regions of the circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1) genes of P. cynomolgi with primers that had been designed to amplify similar regions in P. vivax. These reactions yielded identical PCR products by electrophoresis (CSP=874 bp, MSP-1=839 bp) and by sequencing, from both the initial bloodstream infection and the first relapse due to P. cynomolgi. In contrast, considerable diversity was observed among control P. cynomolgi and P. vivax isolates. These results suggest that the same parasite clones maybe responsible for both initial (primary) and relapse (secondary) infections in P. cynomolgi, and other relapsing malarias.